Koh, Seng Fook (2016) Colony morphotype, biofilm formation and identification of lectins of Burkholderia pseudomallei / Koh Seng Fook. Masters thesis, University of Malaya.
Abstract
Burkholderia pseudomallei, the causative agent of melioidosis, is an important bacterial pathogen in the tropical regions. Melioidosis, the disease caused by B. pseudomallei, has been reported with high mortality and morbidity rates in the endemic regions. Although lectins (sugar binding proteins) had been reported to be important for biofilm formation of several Gram-negative bacteria including Pseudomonas aeruginosa and Burkholderia cenocepacia, there is yet any study on the lectins of B. pseudomallei. This study investigated biofilm production of 76 clinical isolates of B. pseudomallei using a standard biofilm crystal violet staining assay. The results obtained were correlated with their respective colony morphotypes on Burkholderia pseudomallei selective agar medium. As lectin has been reported to initiate bacterial biofilm formation, this study aims to identify, clone and express hypothetical lectin genes in B. pseudomallei. The hypothetical genes were also explored for development of a multiplex polymerase chain reaction (PCR) assay for identification of B. pseudomallei and other closely related species. Based on the colonial morphology of B. pseudomallei on B. pseudomallei selective agar medium, seven distinct colony morphotypes were identified in this study. Most isolates (40.8 %) were identified as colony morphotype group 1 which displayed a rough centre with irregular circumference on the agar medium. Of the 76 B. pseudomallei isolates investigated, 20 (26.3 %) were identified as high biofilm producer (X>11.01), while 37 (48.7%) isolates were medium (3.45<X<11.01), and 19 (25.0 %) were low biofilm (X<3.45) producers, when compared to B. thailandensis ATCC 700388 strain. No correlation was found between B. pseudomallei morphotypes with biofilm forming abilities (p > 0.05). Seven genes encoding hypothetical lectin (BPSS0713, BPSS0767, BPSS1124, BPSS1488, BPSS1649, BPSS2022, and BPSL2056) were retrieved from the genome sequence of B. iv pseudomallei K96243 reference strain. By inclusion of primers targeting 16S rRNA gene and two hypothetical lectin genes (BPSS2022 and BPSS1649), a multiplex PCR assay was successfully developed and evaluated for rapid differentiation of B. pseudomallei, B. mallei, B. thailandensis and B. cepacia complex. The PCR assay was specific and was able to detect up to 109, 60, 23, and 9 ng of the DNA of B. pseudomallei, B. mallei, B. thailandensis and B. cepacia complex, respectively. Four hypothetical genes (BPSS0713, BPSS0767, BPSS1124, and BPSS1488) were successfully cloned and expressed as recombinant proteins in this study. However, none of the recombinant proteins demonstrated positive findings for the hemagglutination assays. Thus, the functions of four hypothetical lectin genes of B. pseudomallei were not confirmed. Many factors including post-translational modification, protein denaturation, and absence of co-factors might affect the expression of lectin activity. For future investigation, glycan array, isothermal titration calorimetry or surface plasmon resonance could be explored to identify lectin in B. pseudomallei.
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